ObjectiveTo observe ovarian microvascular damage in rats with cold coagulation and blood stasis syndrome and to explore the mechanism by which Liangfang Wenjing decoction improves this condition in rats. MethodsFifty SPF female SD rats were randomly divided into a blank group， a model group， low-dose （8.1 g·kg^-1） and high-dose groups （16.2 g·kg^-1） of Liangfang Wenjing decoction， and a 4-phenylbutyric acid （0.1 g·kg^-1） group， with 10 rats in each group. The ice-water bath method was employed to establish the rat model of cold coagulation and blood stasis syndrome. Concurrent with modeling， Liangfang Wenjing decoction was administered continuously for 21 days， once daily. The rats' syndrome manifestations and estrous cycles were recorded. The enzyme-linked immunosorbent assay （ELISA） was used to detect serum reproductive hormone levels and levels of endothelin-1 （ET-1）， nitric oxide （NO）， thrombomodulin （TM）， and von Willebrand factor （vWF） in ovarian tissue. Prothrombin time （PT）， activated partial thromboplastin time （APTT）， thrombin time （TT）， and fibrinogen （FIB） were measured. The ovarian microcirculatory blood perfusion was detected by laser speckle contrast imaging. Hematoxylin-eosin （HE） staining was performed to observe the ovarian histopathology， flow cytometry to detect ovarian apoptosis rate， and transmission electron microscopy to observe the ultrastructure of ovarian microvascular endothelial cells. Western blot was employed to detect the protein expression of endothelial nitric oxide synthase （eNOS）， phosphorylated eNOS （p-eNOS）， Caspase-3， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， glucose-regulated protein 78 （GRP78）， C/EBP homologous protein （CHOP）， inositol-requiring enzyme1α （IRE1α）， p-IRE1α， apoptosis signal-regulating kinase 1 （ASK1）， p-ASK1， c-Jun N-terminal kinase （JNK）， and p-JNK. Immunofluorescence was used to detect ovarian Bax and Bcl-2 expression in microvascular endothelial cells. ResultsCompared with the blank group， the model group showed signs of cold coagulation and blood stasis syndrome， prolonged estrus cycles， and reproductive hormone disorders. Histopathological results revealed a decrease in follicle counts at all stages and disorganized granulosa cell arrangement. Ovarian microcirculatory perfusion was significantly decreased （P<0.01）. PT， APTT， and TT were reduced （P<0.05， P<0.01）， while FIB levels were increased （P<0.05）. In ovarian tissue， NO content was decreased， while ET-1， vWF， and TM levels were increased significantly （P<0.01）. The apoptosis rate of ovarian cells was markedly increased （P<0.01）. Furthermore， p-eNOS/eNOS and Bcl-2 were decreased （P<0.05）， whereas Bax， cleaved-Caspase-3/Caspase-3， GRP78， CHOP， p-IRE1α/IRE1α， p-ASK1/ASK1， and p-JNK/JNK expression showed significant increases （P<0.05， P<0.01）. Compared with the model group， Liangfang Wenjing decoction intervention alleviated the symptoms of cold coagulation and blood stasis， gradually restored the estrus cycle， and improved ovarian histopathology and endothelial cell ultrastructure. Microcirculatory blood perfusion was significantly elevated （P<0.05）. NO content in ovarian tissue was elevated， while ET-1， vWF， and TM levels were significantly decreased （P<0.05， P<0.01）. The p-eNOS/eNOS ratio and Bcl-2 expression were significantly elevated （P<0.05）， while the expression of Bax， cleaved-Caspase-3/Caspase-3， GRP78， CHOP， p-IRE1α/IRE1α， p-ASK1/ASK1， and p-JNK/JNK was significantly decreased （P<0.05， P<0.01）. ConclusionLiangfang Wenjing decoction may regulate the IRE1α/ASK1/JNK signaling pathway to inhibit endoplasmic reticulum stress， attenuate apoptosis， and improve microvascular endothelial injury in ovaries of rats with cold coagulation and blood stasis syndrome.

ObjectiveTo explore the effect and mechanism of Sishenjian on synovial lesions induced by monosodium iodoacetate （MIA） in rats with knee osteoarthritis （KOA）. MethodSixty female Sprague-Dawley （SD） rats were randomly divided into the following six groups： normal group， model group， celecoxib group， and high-， medium-， and low-dose Sishenjian group. The KOA rat model was established by intra-articular injection of MIA. Celecoxib （18 mg·kg^-1） and Sishenjian （14.4， 7.2， 3.6 g·kg^-1） were administered by gavage according to the groups. All rats were euthanized after four weeks of continuous administration. The transverse diameter of the bilateral knee joints of rats was measured， and gross observation of the knee joint was performed. Pathological changes in knee joint synovial tissue were observed by hematoxylin-eosin （HE） staining and picrosirius red staining. Immunohistochemistry （IHC） was used to detect the expression of vascular endothelial growth factor A （VEGFA） in synovial tissue. The levels of inflammatory cytokines in the joint synovial fluid were detected by enzyme-linked immunosorbent assay （ELISA）. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the expression of mRNA and proteins related to the transforming growth factor-β₁ （TGF-β₁）/Smad2/3 pathway in knee joint synovium. ResultCompared with the normal group， the transverse diameter of the knee joint in the model group significantly increased （P<0.01）. Compared with the model group， the transverse diameter of the knee joint in rats of each Sishenjian group significantly decreased （P<0.01）. Compared with the normal group， the expression levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the knee joint synovial fluid of model group significantly increased （P<0.01）. Compared with the model group， the expression levels of IL-1β and TNF-α in the knee joint synovial fluid of rats in each Sishenjian group significantly decreased （P<0.01）. Compared with the normal group， the expression levels of TGF-β₁， Smad2/3， phosphorylation（p）-Smad2/3， type Ⅰ collagen α1 （ColⅠ_α1）， type Ⅲ collagen α1 （ColⅢ_α1）， VEGFA proteins and TGF-β₁， Smad2/3， ColⅠ_α1， ColⅢ_α1 mRNA in knee joint synovium of model group significantly increased （P<0.01）. Compared with the model group， the expression levels of TGF-β₁， Smad2/3， phosphorylation （p）-Smad2/3， ColⅠ_α1， ColⅢ_α1， VEGFA proteins and TGF-β₁， Smad2/3， ColⅠ_α1， ColⅢ_α1 mRNA in knee joint synovium of rats in each Sishenjian group significantly decreased （P<0.05， P<0.01）. ConclusionSishenjian can inhibit synovial inflammation and angiogenesis， and may become a potential drug for treating synovial lesions in KOA by regulating the TGF-β₁/Smad2/3 pathway.

Objective:To observe the dynamic changes of the temporomandibular joint (TMJ) disc in joint movement under different Angle′s classification, providing reference for understanding joint functional movement and providing a basis for more accurate clinical imaging diagnosis.Methods:A total of 30 patients (13 males and 17 females) with temporomandibular disorders who were admitted to Beijing Friendship Hospital, Capital Medical University and General Hospital of the People′s Liberation Army from January 2022 to April 2024 were enrolled. Thirty adults (13 males and 17 females) with different Angle′s classification, with an average age of (34.4±8.5) years, were subjected to dynamic imaging of their TMJ from the closed position to the maximum opening position, and then to the closed position using MRI. The position and morphological changes of the articular discs were observed.Results:The results showed that volunteers with no displacement of the articular disc in class Ⅰ, Ⅱ, and Ⅲ relationships had different shapes of the articular disc during open and closed mouth movements. However, in the maximum opening position, the articular disc were all located directly below the maxillary nodules, and their shape is double concave. In terms of irreversible anterior displacement of the articular disc, in class Ⅰ Angle, the posterior zone of the disc contacts the anterior inclined plane of condyle from the maximum opening position back to the front of the closing position. In class Ⅱ, the posterior zone of the disc contacts the anterior inclined plane of condyle from the beginning of opening position to maximum opening position. In class Ⅲ, the posterior zone of the disc is always in contact with the anterior inclined plane of condyle throughout the entire movement process. And among them, the articular disc presents a forward displacement state at the closing position, its morphology undergoes folding phenomenon. When the openness is 2.5 cm, the articular disc moves up to a certain extent, and is closer to the anterior inclined plane of condyle, and its shape is also partially changed. When the openness is 4.3 cm, the shape of the articular disc, located between the anterior inclined plane of the joint node and the posterior inclined plane of the condyle, is typical double concave, which is sufficient to show that the articular disc is reversible when maximum opening position is reached. In terms of reversible anterior disc displacement, in class Ⅰ Angle, the posterior zone of the disc contact with the anterior inclined plane of condyle at the beginning of the opening position and the end of the closing position. In classⅡ Angle, the posterior zone of the articular disc is not in contact with the anterior inclined plane of condyle. In class Ⅲ Angle, the posterior zone of the articular disc contact with the anterior inclined plane of condyle at the end of the closing position.Conclusions:Multi level dynamic MR imaging data of the temporomandibular joint can dynamically observe the movement of the temporomandibular joint, intuitively and accurately display the position and shape of the articular disc during movement, and can serve as a useful supplement to static conventional MR imaging of the TMJ. The patient's TMJ needs to reach the maximum opening position in order to determine whether the joint disc displacement can be reversible or not.

AIM: To investigate the protective effect of eugenol against Fusarium solani(F.solani)-induced fungal keratitis(FK)in mice and to preliminarily explore possible underlying mechanisms.METHODS: A modified epifluorescence microscopy method was used to prepare the FK mouse model. An equal amount of DMSO(0.05%)was applied to the conjunctiva of the right eye of rats in the dimethyl sulfoxide(DMSO)group. The eugenol group was prepared by applying eugenol(160 μg/mL)to the conjunctival sac of the right eye of mice. The insulin-like growth factor-1(IGF-1)group was coated with the PI3K/AKT pathway activator IGF-1(10 nmol/mL)in the conjunctival sac of the right eye in addition to the administration of eugenol. The corneal morphology was observed under a slit-lamp microscope on days 1, 3, and 5 of inoculation with F.solani suspension, respectively. Hematoxylin eosin(HE)staining was used to assess corneal histopathological damage. The bacterial load of corneal tissue was determined. Enzyme-linked immunosorbent assay and Western blot were used to analyze the levels of inflammatory mediators interleukin-6(IL-6)and interleukin-1β(IL-1β)and the expression of PI3K/AKT pathway proteins.RESULTS: Eugenol treatment improved the morphological symptoms of keratitis and inflammatory response in FK mice, and reduced corneal pathologic tissue damage and fungal load. At 3 d after F.solani infection, corneal tissue IL-6 levels were significantly higher and IL-1β levels were significantly lower in the eugenol group compared with the DMSO group(both P<0.05); corneal tissue IL-6 levels were significantly higher and IL-1β levels were significantly lower in the eugenol group than in the IGF-1 group(both P<0.05). At 5 d after infection, both IL-6 and IL-1β levels in corneal tissue of the eugenol group were significantly lower than those of the DMSO and IGF-1 groups(P<0.05); compared with the DMSO group, the expression of p-PI3K and p-Akt in the corneal tissues of the eugenol group was significantly reduced(P<0.05); the expression of p-PI3K and p-Akt in corneal tissues was significantly lower in the eugenol group than that of the IGF-1 group(both P<0.05).CONCLUSION: Eugenol may attenuate F.solani-induced corneal inflammation by inhibiting the PI3K/AKT pathway, and it has a protective effect against F.solani keratitis in mice.

Objective To establish an improved simple and efficient method for isolation and culture of primary lung fibroblasts(LFB)from neonatal mice,and study their growth characteristics in vitro.Methods The lungs of 3-days-old mice or 8-weeks-old mice were taken under sterile conditions,and the stromal tissue was cut into 1mm3 tissue mass.High-glucose DMEM culture medium containing 10g/dl fetal bovine serum(FBS)or 20g/dl FBS was used for tissue adhesion culture.The LFB was purified by the differential time adhesion method,and the growth morphology and adherence state of the cells were observed dynamically under an inverted microscope.The primary LFB was identified by flow cytometry.The activity of the third-generation cell after culture was detected by CCK-8 assay.Results Lung tissues in mice at 3 days of age and 20g/dl FBS concentration cultured by the improved tissue mass adherent cultured method began to grow radially to the periphery on the 2nd day.On the day 7th,the cells growth density reached 90%,and the cell morphology was as a spindle.The CD140a positive and CD45 negative cells reached more than 90%after purification by differential time adhesion method,and the cells still maintain good cell activity after cultured for 3 generations.Conclusion The improved tissue adhesion method can obtain a large number of purified mice LFB with good activity simply and efficiently,which lays a foundation for the study of lung inflammation,tumors and in vitro efficacy of drugs.

Objective To investigate the role and related mechanism of the soluble guanylate cyclase(sGC)-cGMP-protein kinase G(PKG)signaling pathway in the amelioration of isoproterenol(ISO)-induced cardiac hypertrophy in mice by aqueous extract of Curcuma kwangsiensis root tubers(GYJS).Methods 72 KM male mice were divided randomly into 6 groups:normal,model,propranolol control(40 mg/kg),and GYJS low-(1 g/kg),medium-(2 g/kg),and high-dose(4 g/kg)groups.Mice in the experimental groups were injected subcutaneously with ISO 10 mg/kg on days 1～3 and ISO 5 mg/kg on days 4～14 to establish a mouse cardiac hypertrophy model.4h after each subcutaneous injection of ISO,the mice in each group were administered the corresponding drugs orally for a dosing cycle of 14 days.The hearts were then removed and whole heart and left ventricle weight were measured.Myocardial tissue pathology was observed using hematoxylin and eosin and Masson staining,and sGC subunit beta-1(GUCY1B3)and transforming growth factor(TGF-β1)were detected by immunohistochemistry.Serum lactate dehydrogenase(LDH),creatine kinase(CK),and Nitric Oxide(NO),and myocardial superoxide dismutase(SOD)activity and malondialdehyde(MDA)content were measured using respective kits.Serum cGMP was detected by enzyme-linked immunosorbent assay and quantitative reverse transcription PCR(RT-qPCR),and atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),GUCY1B3,PKG Ⅰ,and phosphodiesterase(PDE)5A mRNA expression levels were also determined by RT-qPCR.Results Compared with the model group,whole heart and left ventricle weights were significantly reduced in mice treated with propranolol or GYJS(P＜0.001 or P＜0.0001)and myocardial hypertrophy and myocardial fibrosis were significantly reversed.All the treatments significantly elevated myocardial expression of GUCY1B3(P＜0.05 or P＜0.01)and significantly reduced expression of TGF-β1(P＜0.05 or P＜0.01).The myocardial damage markers LDH and CK were significantly reduced(P＜0.05 or P＜0.01)while NO and cGMP were significantly elevated(P＜0.05 or P＜0.01),the myocardial oxidative stress indicator MDA was significantly reduced(P＜0.05 or P＜0.01)and SOD activity was significantly increased(P＜0.05 or P＜0.01).mRNA levels of the myocardial hypertrophy markers ANP,BNP,and PDE5A were significantly reduced(P＜0.05,P＜0.01,or P＜0.001)and the mRNA levels of GUCY1B3 and PKG Ⅰ were significantly increased(P＜0.01 or P＜0.001).Conclusions GYJS may improve cardiac hypertrophy by modulating the sGC-cGMP-PKG signaling pathway.

Objective To investigate the effects of insulin therapy on the mechanical behavior of solids,characteristics of fluid flow,and bone marrow stromal cells(BMSCs)differentiation in the distal femoral cancellous bone of type 2 diabetic rats under normal activity and vigorous exercise conditions.Methods The finite element models of cancellous bones and fluids in the distal femurs of rats in the control,diabetes,treatment,and placebo groups in 4-week and 8-week insulin treatment experiments under normal activity and vigorous exercise conditions were established based on micro-CT scanning images.The mechanical and cell differentiation parameters of the models in each group were analyzed using the fluid-solid interaction numerical simulation method.Correlations between mechanical,cell differentiation,and microstructural morphology parameters were also analyzed.Results Insulin therapy under normal activity and vigorous exercise conditions improved the solid and fluid mechanical parameters and BMSC differentiation parameters in type 2 diabetic rats.In the 4-week experiment,insulin treatment under normal activity and vigorous exercise conditions increased the differentiation areas of bone in type 2 diabetic rats from 64.024%to 69.372%and from 73.225%to 75.336%,respectively;in the 8-week experiment,insulin treatment under normal activity and vigorous exercise conditions increased the differentiation areas of bone in type 2 diabetic rats from 67.239%to 72.910%and from 76.147%to 78.291%,respectively.Morphological parameters BV/TV,Tb.N,Tb.Th,Tb.Sp,and structure model index were significantly correlated with the differentiation areas of the bone and cartilage(P<0.05).Conclusions Under vigorous exercise conditions,BMSCs on the surface of cancellous bone in the 8-week insulin treatment group were more likely to differentiate into bone tissue.This study is of great significance for further understanding the effects of insulin on the bone under normal activity and vigorous exercise conditions,and provides theoretical guidance for the selection of the insulin therapy cycle and exercise mode in the clinical treatment of type 2 diabetes.

Objective To investigate the clinical manifestations,molecular genetics and gonadal pathol-ogy characteristics of patients with disorders of sex development(DSD),and to summarize the clinical experi-ence of identifying rare diseases from common symptoms.Methods The clinical data of 416 patients with DSD diagnosed and treated in the multidisciplinary center of DSD of Guangzhou Women and Children's Medical Cen-ter from May 2018 to August 2023 were retrospectively analyzed,summarized and discussed.Results Accord-ing to chromosome karyotype,416 cases of DSD were classified into three types:92 cases(22.1％)of abnormal sex chromosome karyotype,285 cases(68.5％)of 46,XY karyotype and 39 cases(9.4％)of 46,XX karyotype.Among the 92 patients with abnormal sex chromosome karyotype,59 cases were raised as males,18 cases(30.5％)complained of short penis with hypospadias and cryptorchidism.The most common karyotype was 45,X/46,XY(58 cases,63.0％).Among the 285 patients with 46,XY karyotype,238 cases were raised as males,and 63 cases(26.5％)complained of short penis and hypospadias;47 cases were raised as females,and 13 ca-ses(27.7％)complained of inguinal mass.A total of 216 patients with 46,XY karyotype were subjected to whole exome gene detection,and 155 cases(71.8％)were found to have molecular pathogenesis with the clinical phe-notype.Among the 39 patients with 46,XX karyotype,19 cases were raised as males,and 8 cases(42.1％)com-plained of short penis and hypospadias.In the 18 cases of gonad biopsy,17 cases showed testicular tissue in go-nads.Whole exome sequencing was performed in 14 cases.NR5A1 gene heterozygous mutation,SRY gene muta-tion and SOX3 gene mutation were found in 2 cases,respectively(14.3％).Twenty cases were raised as females,and 14 cases(70.0％)complained of clitoral hypertrophy.Gonad biopsy was performed in 8 cases,with 7 cases of ovotestis(87.5％)and 1 case of NR5A1 gene heterozygous mutation(14.3％).Conclusions The etiologies of DSD are complex and diverse,and the clinical manifestations are various,which can be manifested as hypospa-dias,micropenis,cryptorchidism and other common symptoms of the urinary system.Different etiologies have dif-ferent treatment options.Therefore,chromosome karyotype,molecular genetic testing and gonadal pathology can be used to clarify the cause of disease,especially for rare diseases,improve the detection rate,reduce the rate of missed diagnosis,and ensure reasonable treatment,especially sex selection.

Infection is one of the most common causes of death in patients with multiple myeloma, which can lead to early death. The incidence of infection in patients with (multiple myeloma, MM) complicated with diabetes is further increased. Proteasome inhibitors, immunomodulatory drugs and other new drug therapy improve the prognosis of MM, but glucocorticoids used in combination chemotherapy are easy to induce glucocorticoid diabetes, lead to elevated blood glucose, increase the risk of MM infection, and affect the prognosis of patients. This article reviews the relationship between infection and disease progression in patients with MM complicated with diabetes mellitus, infection-related factors, and prevention and treatment measures of different types of pathogen infection.

Objective:To detect the expression of ARK5 in peripheral blood circulating tumor cells (CTCs) from pancreatic cancer patients and explore its correlation with the efficacy and prognosis for gemcitabine chemotherapy.Methods:A total of 175 peripheral blood samples of pancreatic cancer patients who were treated in the Department of Hepatobiliary Surgery, Second Affiliated Hospital of Jiaxing University from January 2016 to June 2021 were collected. CTCs were enriched by nano-microfluidic chip technology. The expression of ARK5 in CTCs was detected by immunofluorescence. According to the expression of ARK5, the patients were divided into two groups: positive group and negative group. The differences on clinicopathological features, the efficacy of chemotherapy, median survival and progression-free survival time between the two groups were compared.Results:CTCs were enriched in 98 of 175 patients (55.6%), including 70 ARK5 positive and 28 ARK5 negative patients. There were no significant differences on clinical features between the two groups, and the two groups were comparable. In the 70 ARK5 positive patients, 64 patients (91.4%) were resistant to gemcitabine, while only 12 of the 28 ARK5 negative patients (42.8%) were resistant to gemcitabine. The incidence of gemcitabine resistance in ARK5 positive patients was significantly higher than that in ARK5 negative patients, and the difference was statistically significant ( P<0.001). The median survival time was 11.5 months in ARK5 positive group and 14 months in ARK5 negative expression group, and the progression-free survival time was 6 months in ARK5 positive expression group and 8 months in negative expression group. The survival time of ARK5 positive group was significantly shorter than that of ARK5 negative group, and the difference was statistically significant ( P<0.05). Conclusions:The pancreatic cancer patients with ARK5 positive CTCs have significantly higher incidence of gemcitabine resistance and shorter survival time than those with ARK5 negative CTCs. Detection of ARK5 expression in CTCs may be a new method to judge chemotherapy efficacy and prognosis for pancreatic cancer patients.